At least four members of the Toll-like receptor (TLR) family recognize some form of nucleic acid. For this strategy of microbial detection to be effective, the immune system must reliably discriminate between self and microbial nucleic acids. Failure to do so can result in autoimmune or auto inflammatory disease. An important aspect of this balance appears to be the intracellular sequestration of TLR9 and TLR7 in endolysosomal compartments. This proposal is the revision of a renewal submission of a grant examining how the cell biology (i.e., localization and trafficking) of TLR9 and TLR7 is regulated and how this regulation contributes to self/non-self-discrimination. In the previous funding period we made three important contributions to this area of research. First, we defined the route of trafficking by which nucleic acid sensing TLRs exit the endoplasmic reticulum and transit to the endolysosome. Second, we described a surprising requirement for ectodomain proteolysis in the activation of TLR9, TLR7, and TLR3. Third, we used a TLR9 mutant to demonstrate the importance of localization and proteolytic regulation in self/non-self-discrimination. Overall, thi work transformed the model for TLR trafficking and regulation and convincingly demonstrated that studying these questions has relevance for self/non-self-discrimination. In this renewal application, we propose to extend these studies to primary cells and in vivo models. In Aim 1, we will use TLR9-HA knock-in mice to study the trafficking and processing of TLR9 expressed at endogenous levels in a range of cell types. In Aim 2, we will determine how Unc93b1 controls the trafficking of endosomal TLRs. In Aim 3, we will use knock-in mice with an inducible allele of a TLR9 mutant that fails to maintain tolerance to self-nucleic acid. These mice will be used to explore the cell types and triggers required for breaking tolerance to self-nucleic acid.